This project will complete the identification and immunogenicity analyses of all proteins encoded by the Mycobacterium tuberculosis (Mtb) genome, yielding a pool of protective antigens that will immediately enter development as subunit vaccines for tuberculosis. In our prior SBIR Phase I grant (R43 AI053636-01), we developed a platform technology for high throughput, proteome-based identification of antigens that have a high probability of inducing protective cellular immunity when formulated and administered as a vaccine. The high throughput process that was developed combines efficient gene amplification and protein expression methods with serum- and splenocyte-based assays to determine the level of antibody- and T-cell specific reactivity against each individualprotein. Using funding provided by our Phase I award, and in collaboration with recognized Mtb vaccine leaders, we have validated this proprietary antigen discovery system, and have successfully completed immunogenicity analysis of 384 genes (approximately 10%) of the Mtb genome. We now seek support to apply this process to the remaining approximately 90% of the Mtb genome to generate a portfolio of candidate antigens for use in vaccine (and diagnostics where appropriate) development, and to complete initial development, production and pre-clinical testing of tuberculosis vaccine candidates based on the identified antigens by our collaborators at Aeras Global TB Vaccine Foundation. Central to our rapid antigen discovery process is our patented gene amplification technology, called Transciptionally Active PCR (TAPR), a cloning-free method developed under a Phase I SBIR grant (R43 AI47641-01) that generates transcriptionally active PCR fragments that can 1) be used to express proteins in cultured cells, 2) be used to directly vaccinate animals (genetic vaccination), and 3) serve as templates to direct cell-free in vitro transcription and translation reactions that yield large amounts of proteins for use in immunoassays. Because it is cloning-free, TAP is a powerful tool for rapid synthesis and amplification of both genomes and the corresponding proteomes, and coupled with B- and T-cell immunoassays serve as a high-throughput antigen discovery platform called Vaccinomics(TM/SM). To date, Vaccinomics(TM/SM) has been used to rapidly amplify, express, and analyze the immunogenicity of approximately 10% of the Mtb proteome. We now propose to complete this process for the entire Mtb proteome by 1) applying bioinformatic analyses to predict the immunogenicity of each open reading frame (ORF) and rank the genome accordingly, then 2) synthesizing and purifying the proteins to create protein arrays for subsequent immunological screening using material from Mtb-infected mice, guinea pigs, and human TB patients to select vaccine antigen candidates: Vaccine candidates identified by the T cell assays will be systematically evaluated for their immunogenicity and protective immunity in animal models including Mtb-infected mice and guinea pigs. First, C57BL/6 mice will be immunized by vaccine candidates to examine their immunogenicity. Second, the antigens will be further examined for their protective immunity against aerosol TB infection of mice. The leading vaccine candidates identified in mouse studies will be further examined in the more stringent guinea pig model with a prime and boost strategy, and the protective efficacy is directly compared to that of Bacillus Calmette Guerin (BCG). Finally, a selected number of highly promising vaccine candidates will be expressed by recombinant BCG vectors and tested for their protective immunity in guinea pigs under conditions in compliance with clinical study requirements. Based on these studies, promising candidate vaccines will enter advanced development at Aeras in anticipation of expedited clinical testing. We also suggest that this approach will likely be useful for the development of rational vaccines and diagnostics against other naturally emerging and genetically engineered organisms, and may also be particularly useful when rapid responses to novel infectious diseases (ID) and drug-resistant IDs including TB are required.